Magnetic Resonance Imaging is a well established powerful tool for medical and biological investigations both in vitro and in vivo. The main drawback of this technique is due to the intrinsic low sensitivity of the NMR spectroscopy on which MRI is based. In fact, the intensity of NMR signals depends on the difference between the nuclear spin states populations of the imaging nuclei. According to the well known Boltzman equation (ΔN=γhB0/(2πkT)), this difference is a function of temperature and applied magnetic field, and, at thermal equilibrium, it is in the order of 10−5, i.e. very low.
The use of hyperpolarized molecules has been recently proposed as a possible solution of the said drawback and, in recent years, many efforts have been devoted to the development of both feasible and effective MR-hyperpolarization procedures.
In this regard, the most straightforward is the so called “brute force” method, that consists in keeping the molecule of interest at high magnetic field strength (up to 20 T) and low temperature, close to absolute zero, for a given period of time. This method is of general applicability, but it requires the application of an appropriate “relaxation switch” that consents to quickly promote the nuclear transitions needed to create nuclear polarization, and that must be removed or, otherwise, “turned off” immediately after the trial. However, as a “relaxation switch” fit for such a purpose has not already been found, this approach is not, at now, exploitable.
A second method is the so-called “optical pumping/spin exchange”, which can be applied to noble gases such as 129Xe and 3He. In this case a circularly polarized laser beam is used to irradiate a gaseous mixture of the selected gas and an alkaline metal vapour. This allows providing Xe and He with a high degrees of polarization that can be maintained for long time, thanks to the long relaxation times of these nuclei.
MRI studies of the respiratory system conducted with the use of hyperpolarized gases thus obtained are known. In this regard, see, for instance, J. Thoracic Imag. 2004, vol. 19, pp. 250-258; Phys. Med. Biol. 2004, vol. 49 pp. 105-R153. However, as formerly said, this technique is not of general applicability, but it is just limited to the polarization of noble gases.
The population difference between nuclear spin levels can also be increased through exploiting the “Overhauser” effect between the nucleus of interest and the unpaired electrons of coupled paramagnetic species, according to a technique known as Dynamic Nuclear Polarization or DNP. This technique has been used, for instance, to hyperpolarize some molecules of biological interest, including urea, pyruvate and metabolic derivatives thereof, currently exploited for studies of metabolic profiles performed by use of MRI (see, for instance, Europ. Radiol. 2006, vol. 16, pp. 57-67, Magn. Res. in Med., vol 58, 2007, pp. 65-69; Appl. Magn. Res. 2008, vol. 34, pp. 533-544).
Although this technique may be applied, at least in principle, to any type of molecule, the need for a powerful cryostat and a suitable “hardware” allowing the irradiation of electrons at low temperature constitutes, in fact, a limit to its more general use.
Further difficulties may also arise from the need for an efficient procedure allowing the rapid dissolution of the substrate after the hyperpolarization, and the separation of the highly toxic paramagnetic radical before the in vivo administration of the hyperpolarized substrate.
The alternative use of hyperpolarized molecules obtained by addition of para-hydrogen on unsaturated substrates by means of a procedure known as Para Hydrogen Induced Polarization (PHIP) has also been proposed, for instance, in U.S. Pat. No. 6,574,495.
The main advantage of this procedure relies on that it allows to obtain populations of the nuclear spin levels deeply altered compared with those determined by the Boltzmann thermodynamics without having to use extremely low temperatures and complex dissolution procedures such as those used in the DNP method. As such, it can be regarded as a simpler and cheaper alternative to the DNP hyperpolarization technique.
As said, the PHIP procedure relies on the catalytic hydrogenation of an unsaturated substrate, or MR agent precursor, with hydrogen gas enriched in the para isomer.
The hydrogen molecule, in fact, exists in two isomeric spin forms, namely ortho-hydrogen (o-H2), and para-hydrogen (p-H2). The ortho isomer, which is symmetric with respect to the exchange of the two protons, is triply degenerate (triplet state), while the para isomer, which is anti-symmetric, is a singlet state. Furthermore, the ortho isomer has spin equal to 1 (S=1) and is NMR active, while the para isomer, having spin equal to 0 (S=0), is NMR silent.
At room temperature the equilibrium mixture in which the two forms exist, otherwise called normal-hydrogen, contains 75% of ortho and 25% of para isomer, but, being the para state thermodynamically favoured and due to the relatively high rotational temperature of the H2 molecule, it is possible to enrich the equilibrium mixture in the para isomer, by keeping it at low temperature.
For instance, at 77 K (liquid N2 temperature) the two forms exist with a 52% (para): 48% (ortho) ratio and at 20 K the mixture is formed by 99.8% of the para isomer.
At normal conditions, the equilibration rate between the two isomers is very low, because it involves a singlet-triplet transition that is forbidden by selection rules. However, in the presence of a suitable catalyst, for instance selected from iron oxides such as Fe3O4, Fe2O3 and activated charcoal, the interconversion may be rapidly obtained (for example, in few hours). The para enrichment thus obtained can be then maintained at room temperature, provided that the conversion catalyst, and any other paramagnetic impurity, are totally removed. By this way it is possible to have a non-equilibrium mixture, i.e. a hydrogen mixture enriched in the para isomer, also called para-hydrogen, at room temperature, that remains stable for several hours.
Although NMR silent, when para-hydrogen is added to an unsaturated molecule, its symmetry can be broken with the formation of an AX spin system allowing to observe the hyper-polarization, or, in other words, a significantly intensified signal in the NMR spectrum of the para-hydrogenated compound that corresponds to the hyperpolarized proton nucleus. Typically, in 1H NMR spectra the sensitivity increase measured in terms of enhancement of the MR signal can be as high as a factor 105 (see, for instance, Sensitivity enhancement utilizing parahydrogen, C. R. Bowers, Encyclopedia of NMR Vol. 9 2002 pp 750-770).
Nevertheless, a skilled practitioner is aware that, for in vivo MRI purposes, heteronuclear (non-proton) hyperpolarization is more useful than that, even so high, of protons. That is because, in in vivo conditions, the proton signal of a parahydrogenated contrast agent would overlap with endogenous 1H signals of tissue water. On the contrary, the almost total absence of endogenous signal for non-proton nuclei results in the practical absence of background noise, thus allowing for the registration of images with a high signal to noise ratio, where the contrast is only given by the difference in signal intensity between regions reached by the hyperpolarized molecule and the areas in which the same is absent.
Further benefits are due to longer T1 values characterizing non-proton nuclei (which limit the polarization loss due to relaxation) and to the width of chemical shift range associated with the same, when included in different molecules, or, in other words, to the fact that the value of chemical shift associated with a given heteronucleus is different into different molecules, wherein this makes possible to view different molecules at one time.
The main interest is, therefore, directed to feasible and effective MR-procedures for the polarization of non-proton nuclei, especially of nuclei having nuclear spin=½ such as, for instance, 13C, 15N and 29Si, as well as to contrast agents comprising non-proton hyperpolarized nuclei, and, especially, 13C enriched hyperpolarized substances.
Interestingly, the PHIP hyperpolarization method allows to provide 13C and 15N hyperpolarized molecules in a simpler and cheaper way, especially when compared with the DNP technique.
For contrast, in order to obtain a 13C hyperpolarized molecule that is effective for use in in vivo MRI medical imaging, the following requirements must be satisfied:                i) The substrate molecule must be easily hydrogenable;        ii) The substrate molecule must contain a 13C carbon atom within a distance of three bonds from the protons added to the molecule with para-hydrogen;        iii) The molecular weight of the substrate molecule should be low, and preferably lower than 500 Da, in order to limit the relaxation rate;        iv) The hyperpolarization product must be water soluble and physiologically tolerable;        v) A parahydrogenation catalyst must be used allowing to promote the transfer of both protons from one H2 molecule to the same substrate molecule, so that the spin correlation is maintained. Catalysts enabling this kind of transfer are the homogeneous ones, such as, for instance, Rh or Ir organometallic complexes that, due to their high toxicity, must be removed from the reaction mixture before injection;        vi) To be effective in MRI imaging, the spin-order of the para-hydrogen has to be transformed into 13C net magnetization.        vii) As aqueous solutions of the parahydrogenation product are used for in vivo applications, the hydrogenation reaction should be carried out directly in water or, alternatively, the organic solvent being used for hydrogenation must be totally removed.        
It will be apparent to a skilled person that the same kind of criteria equally applies for the preparation of hyperpolarized substances suitably enriched with a non-proton nucleus different from 13C.
It stems from the above that the main problems one has to face when using PHIP hyperpolarization methods are due to the use of toxic hydrogenation catalysts and of organic solvents in which both the hydrogenation catalyst and hydrogen are more soluble, while, for contrast, for in vivo MRI applications physiologically acceptable aqueous solutions of the parahydrogenation products are needed.
Several kinds of hydrogenation catalysts based on transition metals are, therefore, under intense scrutiny.
Homogeneous transition metal based catalyst have shown to offer best activity and selectivity. In particular, catalysts that have shown to allow higher polarization on 13C after para-H2 addition to unsaturated precursor are Rh(I) cationic complexes, preferably containing a chelating phosphine ligand, for example DPPB (diphenylphosphine butane) or DPPE (diphenylphosphine ethane), and a diene molecule such as, for instance, cyclooctadiene or norbornadiene (see, for instance, K. Goldman et al., Magn. Res. Med. 2001, 46 1-5)
The highest efficiency of these hydrogenation catalysts is achieved in organic solvents, preferably in acetone, in which they are more soluble. It is, however, clear that organic solvents must be totally removed from the reaction mixture before the same is formulated in an aqueous medium for the in vivo administration. This task can, for instance, be achieved by means of a “spray-drier” located immediately downstream of the reactor, through a process similar to that commonly used in pharmaceutical technology to transform a solution into solid dry particles (see, for instance, U.S. Pat. No. 3,615,723). In this case, the fluid material is sprayed into the drying chamber where it is nebulised and dispersed by a carrier gas; the more volatile solvent is then distilled by applying vacuum while water, previously added to the mixture, remains in the “drier” thus providing a water solution of the added material. However, as low molecular weight molecules (less than 500 Da) are preferably used as parahydrogenation substrates, a main drawback associated with the use of the above procedure stems from the possible loss of the hydrogenation product along with the organic solvent.
On the other side, aqueous solvents have also been used, for instance in WO99/24080, together with Rh(I) cationic complexes containing some ionic/polar groups, mainly on the phosphyne ligands, purposely introduced to improve the catalysts water solubility and, in turn, their efficiency in an aqueous medium.
However, an important drawback to be addressed when working in an aqueous medium is due to the low solubility of hydrogen in water that makes it necessary to operate at very high pressure (50-100 bar) or at lower pressure (10-15 bar) but under laminar flow conditions and into suitable reactors disclosed, for instance, in Magn. Res. Mater. Phys. 2009, 22, 111. In addition, the use of homogeneous catalyst is hampered by the difficulties of catalyst recovery and recycling with simultaneous isolation of a catalyst-free product solution.
A method typically used to remove cationic Rh complexes comprises, for example, percolating the reaction mixture on a suitable cation-exchange resin, although this procedure leads to a marked loss of polarization.
Catalysts based on Rh (I) supported on a solid surface, for instance silica or polymers, have, alternatively, been used. However, the net polarization obtainable with supported catalysts is significantly lower than that observed with homogeneous catalysts, probably because of the lower mobility of the substrate-catalyst adduct that results in an increased relaxation rate at intermediate level. Therefore, a need still remains for easy and cheap procedures able to overcome the above purification problems and to provide aqueous solutions of hyperpolarized molecules ready for use in MR imaging of a human or non-human animal body.